U.S. patent application number 14/878568 was filed with the patent office on 2016-04-14 for novel ruthenium (ii) complexes, preparation and uses thereof.
The applicant listed for this patent is Council of Scientific and Industrial Research. Invention is credited to Samit Chattopadhyay, Amitava Das, Vadde Ramu, Nandaraj Taye.
Application Number | 20160102357 14/878568 |
Document ID | / |
Family ID | 55655045 |
Filed Date | 2016-04-14 |
United States Patent
Application |
20160102357 |
Kind Code |
A1 |
Das; Amitava ; et
al. |
April 14, 2016 |
NOVEL RUTHENIUM (II) COMPLEXES, PREPARATION AND USES THEREOF
Abstract
The present invention discloses novel Ruthenium (II) polypyridyl
complexes, preparation and its application as DNA imaging
agents.
Inventors: |
Das; Amitava; (Pune, IN)
; Chattopadhyay; Samit; (Pune, IN) ; Ramu;
Vadde; (Pune, IN) ; Taye; Nandaraj; (Pune,
IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Council of Scientific and Industrial Research |
New Delhi |
|
IN |
|
|
Family ID: |
55655045 |
Appl. No.: |
14/878568 |
Filed: |
October 8, 2015 |
Current U.S.
Class: |
435/6.1 ;
544/225 |
Current CPC
Class: |
C07F 15/0053
20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07F 15/00 20060101 C07F015/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 8, 2014 |
IN |
2864/DEL/2014 |
Claims
1. Novel Ruthenium (II) polypyridyl complexes of formula I
##STR00011## wherein, X is selected from hydrogen or fluorine.
2. The novel Ruthenium (II) polypyridyl complexes of formula I,
wherein, the complexes are selected from a)
{bis-(2,2'-bpy)-(5-fluoro-1-((4'-methyl-[2,2'-bipyridin]-4-yl)methyl)pyri-
midine-2,4(1H,3H)-dione)}ruthenium(II) dichloride; and b)
{bis-(2,2'-bpy)-(1-((4'-methyl-[2,2'-bipyridin]-4-yl)methyl)pyrimidine-2,-
4(1H,3H)-dione)}ruthenium(II) dichloride.
3. A process for the preparation of novel Ruthenium(II) polypyridyl
complexes of formula I, ##STR00012## wherein, X is selected from
hydrogen or fluorine comprising; a) reacting
[Ru(bpy).sub.2Cl.sub.2].sup.2+ compound of formula 4 with the
ligand of formula 3 in ethanol; ##STR00013## wherein, X is selected
from hydrogen or fluorine, b) precipitating the Ruthenium(II)
polypyridyl complexes of formula I by the addition of saturated
aqueous potassium hexafluorophosphate (KPF.sub.6) solution to
obtain the desired complex; and c) purifying the desired complexes
of formula I.
4. The process according to claim 3, wherein, the ligand of formula
3 is selected from a)
5-fluoro-1-((4'-methyl-[2,2'-bipyridin]-4-yl)methyl)pyrimidine-2,4(1H,3H)-
-dione and b)
1-((4'-methyl-[2,2'-bipyridin]-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione.
5. The process according to claim 3, wherein, the ligand of formula
3 is prepared by a process comprising reacting
4-(bromomethyl)-4'-methyl-2,2'-bipyridine with 5-fluorouracil or
Uracil in presence of K.sub.2CO.sub.3 and potassium iodide in DMSO
to obtain the
5-fluoro-1-((4'-methyl-[2,2'-bipyridin]-4-yl)methyl)pyrimidine-2,4(1H,3H)-
-dione and
1-((4'-methyl-[2,2'-bipyridin]-4-yl)methyl)pyrimidine-2,4(1H,3H-
)-dione, respectively.
6. A composition comprising Ruthenium (II) polypyridyl complexes of
formula I along with one or more pharmaceutical carriers for use as
DNA imaging agent, ##STR00014## wherein, X is selected from
hydrogen or fluorine.
7. A method of imaging DNA in a tumor/cancer cell lines comprising
(a) Addition of Ruthenium (II) polypyridyl complexes of formula I
as imaging agent; and ##STR00015## wherein, X is selected from
hydrogen or fluorine, (b) subjecting/exposing the mammalian cancer
cell lines to an energy source (.lamda.=442 nm); and (c) observing
the image of DNA by intracellular fluorescence intensities of the
imaging agent or detecting an emission from the imaging agent using
the energy source.
8. The method according to claim 7, wherein, the energy source may
be selected from photon emission computed spectroscopy; positron
emission tomography (PET) and the like.
9. The method according to claim 7, wherein the cancer/tumor cell
may be selected from breast cancer; epithelial cancer, lung cancer,
ovarian carcinoma, pancreatic carcinoma, prostate cancer or
colorectal carcinoma.
10. Ruthenium (II) polypyridyl complexes of formula I, for use as
DNA imaging agents. ##STR00016## wherein, X is selected from
hydrogen or fluorine.
Description
CLAIM OF PRIORITY
[0001] This application claims the benefit of priority of India
Patent Application Serial No. 2864/DEL/2014, entitled "NOVEL
RUTHENIUM (II) COMPLEXES, PREPARATION AND USES THEREOF," filed on 8
Oct. 2014, the benefit of priority of which is claimed hereby, and
which is incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to novel Ruthenium (II)
polypyridyl complexes, preparation and its application as DNA
imaging agents in PFA fixed MCF-7 cells.
BACKGROUND AND PRIOR ART OF THE INVENTION
[0003] The development of molecular probes for selective DNA
imaging is of great importance for studies in cell biology and
clinical diagnosis. The commercially available DNA-specific dyes
generally suffer from the poor water solubility and photo bleaching
issues. In addition, dyes like DAPI
(4',6-diamidino-2-phenylindole), and Hoechst have shorter life
times, small stoke's shift value and require ultra-violet light
illumination. Apart from these, auto fluorescence coming from the
endogenous flourophores (mitochondria, DNA and NADPH) also limit
practical application of such dyes.
[0004] In this regard, Ru (II) polypyridyl complexes offer
excellent luminescent properties and rich photochemistry. Their
high photostablity, larger stoke's shift value (more than 150 nm)
and solubility in aqueous environment makes such derivatives an
ideal candidate for use as cellular imaging agent. Recently, Ru(II)
complexes were used for imaging the structure of the DNA in live
cells.
[0005] An article titled "A ruthenium(II) polypyridyl complex for
direct imaging of DNA structure in living cells" published in
NATURE CHEMISTRY, DOI: 10.1038/NCHEM.406, published in October 2009
discloses dinuclear Ru(II) complexes
[(phen).sub.2Ru(tpphz)Ru(phen).sub.2]4.sup.+ and [(bpy).sub.2
Ru(tpphz)Ru(bpy)2]4.sup.+ wherein, phen=1,10-phenanthroline,
tpphz=tetrapyrido[3,2-a: 2',3'-c:3'', 2''-h:2''', 3'''-j]phenazine,
as shown below, for use as DNA imaging agents with both
luminescence and transition electron microscopy. However, these
complexes are appear to be more of hydrophilic rather than
lipophilic, limiting its cell diffusion across the membrane at low
concentrations and therefore requires relatively in higher
concentration for efficient uptake.
##STR00001##
[0006] Another article titled "Binuclear ruthenium(II) polypyridyl
complexes: DNA cleavage and mitochondria mediated apoptosis
induction" published in Polyhedron, Volume 36, Issue 1, 4 Apr.
2012, Pages 45-55 discloses binuclear complexes of the type
[Ru2(N--N)4(TBPhen2)]4+, where N--N=2,2'-bipyridine (bpy) (1),
1,10-phenanthroline (phen) (2), dipyrido[3,2-a:2',3'-c]phenazine
(dppz) (3) and (TBPhen2)=bis-phenanthroline Troger's Base analogue.
These complexes 1& 2 are found to induce apoptosis. Therefore,
the complexes studied in this article are cytotoxic and hence are
not useful for DNA imaging in cells.
##STR00002##
[0007] Another article entitled "Chiral Ruthenium (II) Polypyridyl
Complexes: Stabilization of G-Quadruplex DNA, Inhibition of
Telomerase Activity and Cellular Uptake" published in PLOS ONE, on
December 2012, Volume 7, Issue 12, e50902, discloses Two
ruthenium(II) complexes, L-[Ru(phen)2(p-HPIP)]2+ and
D-[Ru(phen)2(p-HPIP)]2+ as telomerase inhibitors. These complexes
are reported to have relatively higher selectivity to cancer cells
than to normal cells.
##STR00003##
[0008] Further, most of the reported Ru(II) complexes are
positively charged and possess limited cell membrane permeability.
This significantly restricts their potential application as
cellular imaging agents. To overcome this issue of cell
permeability, the common practice is of using serum free media,
detergent (TritonX-100) or organic solvents such as DMSO, to make
the cellular uptake more facile.
[0009] Recent reports demonstrated that polyarginine conjugated
Ru(II) complexes can be targeted to the nucleus with the help of
long chain polyarginine peptide. However, the preparation of
polyarginine conjugated Ru(II) complexes not only involves much
more synthetic effort, but also requires more incubation time.
Also, the conjugation of polyarginine moiety may enhance the
hydrophobicity of the Ru(II) complexes, thereby limiting their
uptake to the lipid bilayer of the cell membrane. Some dinuclear
Ru(II)polypyridyl complexes were also reported for cellular imaging
but displays relatively poor solubility in pure water. Accordingly,
DMSO has to be employed to prepare working stock solutions.
[0010] If the transition metal complexes need to be functioned as
DNA imaging agents, it is required to possess low cytotoxicity and
high membrane permeability in addition to high solubility and photo
stability. From the above, it is evident that it is difficult to
design such transition complexes that meet the requirement of the
aforementioned properties.
[0011] Therefore there is a pronounced interest in the development
of DNA specific probes which exhibits high water solubility, high
photostablity, high membrane permeability, low toxicity and large
stokes shifts to facilitate its application.
OBJECTIVE OF THE INVENTION
[0012] The main objective of the present invention is to provide
novel Ruthenium(II) polypyridyl complexes that perform as efficient
cellular DNA imaging probes in PFA fixed MCF-7 cells.
[0013] Another object of the invention is to provide a simple
process for the preparation of Ru(II) polypryidyl complexes.
SUMMARY OF THE INVENTION
[0014] In accordance with the above objectives, the present
invention provides novel Ruthenium (II) polypyridyl complexes of
formula 1 as shown below.
##STR00004##
wherein, X is selected from hydrogen or fluorine.
[0015] Accordingly, in an aspect, the present invention encompasses
novel Ruthenium (II) polypyridyl complexes of Formula 1 and 2.
##STR00005##
[0016] In another aspect, the invention provide a one step process
for the synthesis of Ruthenium (II) polypyridyl complexes of
Formula 1 and 2.
[0017] In another aspect, the properties of the Ruthenium (II)
polypyridyl complexes of formula 1 & 2 are found to be highly
water soluble, have large stokes shift (150-170 nm, relatively
low-toxic (<350 .mu.M), appreciably long lifetime (275-400 ns in
aerated aqueous solution) and both excitation and fluorescence in
visible region of the spectrum.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0019] FIG. 1. ORTEP drawing of complex 2: Thermal ellipsoids set
to 50% probability level.
[0020] FIG. 2. Fluorescence life time decay for the complex 1(A)
2(B) and 3(C) fitted with single exponential. Time correlated
single photon counting studies (TCSPC) were performed in aqueous
medium. Excitation source: 443 nm laser.
[0021] FIG. 3. .sup.1H NMR for the ligand L.sub.1 recorded in
chloroform-d.sub.3, the solvent peak was highlighted in blue
circle.
[0022] FIG. 4. .sup.19F NMR for the ligand L.sub.1 recorded in
acetonitrile-d.sub.3.
[0023] FIG. 5. ESI mass spectrum for the ligand L.sub.1.
[0024] FIG. 6. .sup.1H NMR for the complex 1 recorded in
acetonitrile-d.sub.3
[0025] FIG. 7. .sup.19F NMR for the complex 1 recorded in
acetonitrile-d.sub.3
[0026] FIG. 8. ESI-HRMS for the complex 1.
[0027] FIG. 9. .sup.1H NMR spectrum of the ligand L.sub.2.
[0028] FIG. 10. ESI mass spectrum for the ligand L.sub.2.
[0029] FIG. 11. .sup.1H NMR spectrum for complex 2 recorded in
acetonitrile-d.sub.3.
[0030] FIG. 12. ESI-HRMS for complex 2.
[0031] FIG. 13. Cell viability assay of 1 and 2 on MCF-7 and
HEK293T cell line for 24 h incubation.
[0032] FIG. 14. Confocal image of MCF-7 cells incubated with
complex 1(A) and 2(B) for 1 hr.
[0033] FIG. 15. Confocal image of MCF-7 cells incubated with
complex 1(A) and 2(B) for 1.5 hr.
[0034] FIG. 16. HEK 293T cells incubated with complexes 1 (A), and
2(B) for 2 h.
[0035] FIG. 17. CLSM images of MCF-7 cells incubated with complexes
1(A and B), 2(C and D) after treatment with RNase (A, C) and DNase
(B, D).
[0036] FIG. 18. CLSM image that shows cellular uptake and nuclear
staining of MCF-7 cell s incubated for 2 h with 1 (20 .mu.M) (Fig.
A) and 2 (20 .mu.M) (Fig. B) in serum containing media. The exact
overlap of the two fluorescence profile represents that 1, 2 and
DAPI are present at the cell nucleus. Intensity profiles plotted
using ImageJ software.
[0037] FIG. 19. CLSM images that shows the live cell uptake (A) and
fixed cell staining (B) of 1 and 2. Enlarged view (C) of the live
MCF-7 cells incubated with ER tracker green and complexes 1 and 2.
Co-localization studies performed with ER-Tracker.TM. Green for
live cells and with DAPI for fixed cells.
[0038] FIG. 20 Absorbance and emission spectra for 1 (blue line)
and 2 (red dotted line).
[0039] FIG. 21. ITC binding profile for the interaction of 1
(left), 2 (centered)) and 3 (right) with CT-DNA in Tris-HCl buffer
pH=7.4 at 25.degree. C.
[0040] FIG. 22. a) CLSM images of fixed MCF-7 cells stained with 3
for 4 h b) Live MCF-7 cells stained with 3
[0041] FIG. 23. Live MCF-7 cells incubated with complexes 1 and 2
at 4.degree. C.
[0042] FIG. 24. PFA fixed MCF-7 cells incubated with 1 and 2
without DAPI.
DETAILED DESCRIPTION OF THE INVENTION
[0043] The invention will now be described in detail in connection
with certain preferred and optional embodiments, so that various
aspects thereof may be more fully understood and appreciated.
[0044] Accordingly, the present invention provides novel Ruthenium
(II) polypyridyl complexes, preparation and uses thereof.
[0045] In an embodiment the present invention provides novel
Ruthenium (II) polypyridyl complexes of formula I as depicted
below.
##STR00006##
Wherein, X is selected from hydrogen and fluorine.
[0046] Accordingly, the present invention encompasses novel
Ruthenium (II) polypyridyl complexes of formula 1 and formula
2.
##STR00007##
[0047] In another embodiment the present invention provides a
single step process for the preparation of novel Ruthenium(II)
polypyridyl complexes of formula I from novel ligand of formula
3,
##STR00008## [0048] Wherein, X is selected from F or H,
Comprising:
[0048] [0049] a) Reacting [Ru(bpy).sub.2Cl.sub.2].sup.2+ compound
of formula 4 with the ligand 3 in ethanol and [0050] b)
precipitating the desired Ruthenium(II) polypyridyl complexes of
formula I by the addition of saturated aqueous potassium
hexafluorophosphate (KPF.sub.6) solution and [0051] c) purifying
the desired complexes of formula I.
[0052] The above synthetic route followed for the preparation of
complexes of formula 1 and 2 is depicted below in Scheme 1.
##STR00009##
Wherein, X is selected from F(complex 1) or H(complex 2).
[0053] In still yet another embodiment, the present invention
provides novel Ruthenium (II) polypyridyl complexes of formula 1
(X.dbd.F) and formula 2 (X.dbd.H), wherein the said complexes are
highly water soluble, relatively non-toxic, higher .sup.3MLCT
excited state lifetimes, exhibit absorbance (455 nm) and
fluorescence (617 nm) maxima in visible region and possess large
Stoke's shift (165 nm) compared to the traditional DNA staining
agents. Therefore, the present invention provides novel Ruthenium
(II) polypyridyl complexes of formula 1 and formula 2 wherein the
said complexes are used for cellular DNA imaging.
[0054] For the purpose of the present invention, complex of formula
1 is chemically termed as
{bis-(2,2'-bpy)-(5-fluoro-1-((4'-methyl-[2,2'-bipyridin]-4-yl)methyl)pyri-
midine-2,4(1H,3H)-dione)}ruthenium(II) dichloride and complex of
formula 2 is chemically termed as
{bis-(2,2'-bpy)-(1-((4'-methyl-[2,2'-bipyridin]-4-yl)methyl)pyrimidine-2,-
4(1H,3H)-dione)}ruthenium(II) dichloride.
[0055] In another aspect the present invention provides a process
for preparation of ligand 3, wherein compound of ligand 3 is
selected from: [0056] a. Ligand L.sub.1:
5-fluoro-1-((4'-methyl-[2,2'-bipyridin]-4-yl)methyl)pyrimidine-2,4(1H,3H)-
-dione [0057] b. Ligand L.sub.2:
1-((4'-methyl-[2,2'-bipyridin]-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione.
[0058] c. Ligand L.sub.3: 4,4'-dimethyl-2,2'-bipyridine.
[0059] Accordingly, the invention provides a process for
preparation of Ligand L.sub.1 which comprises reacting
4-(bromomethyl)-4'-methyl-2,2'-bipyridine with 5-fluorouracil in
presence of K.sub.2CO.sub.3 and KI in DMSO. Similarly, ligand
L.sub.2 is prepared by reacting
4-(bromomethyl)-4'-methyl-2,2'-bipyridine with Uracil in presence
of K.sub.2CO.sub.3 and KI in DMSO. The ligands 1 & 2 are used
for the preparation of complexes 1 & 2 of the invention.
[0060] In yet another aspect, a model complex 3 is prepared for
comparing with the complexes 1 & 2. The model complex 3,
([Ru(bpy)2(L3)]2+; wherein, bpyis 2,2'-bipyridine &
L3=4,4'-dimethyl-2,2'-bipyridine, as shown below.
##STR00010##
[0061] In yet another embodiment, Cl.sup.- ion is selected as
counter anions for complexes 1, 2 & 3, to achieve the desired
solubility in aq. buffer (Tris-HCl buffer, pH=7.4) media.
[0062] In another aspect, the structural elucidation of complexes
1, 2 & 3 are confirmed by single crystal X-ray structural
analysis, .sup.1H NMR, .sup.19F NMR, EI mass spectrum etc.
[0063] Photophysical properties of the complexes 1, 2 recorded in
pure aqueous medium are provided in table 3. Relative binding
affinity of complexes 1, 2, and 3 towards calf-thymus DNA (CT-DNA)
were evaluated using isothermal titration calorimetry (ITC) (FIG.
20). Association constants and thermodynamic parameters (Table 4)
clearly reveal that the binding affinities for complexes 1 and
2
[0064] are much higher than that of model complex 3 towards CT-DNA.
The binding affinity of all three complexes for the CT-DNA is
1>2>3.
[0065] In yet another embodiment, the invention provides evaluation
of both the complexes 1 and 2 for their cytotoxicity, in order to
extend its application as DNA imaging agents. The cytotoxicity
studies confirm that both the complexes 1 & 2 showed
insignificant cytotoxicity and a lipophilicity dependent cellular
internalization process. Furthermore, when studied in MCF-7 cells,
localization of complexes 1 and 2 were observed only in nucleus and
such observation is hitherto unknown and unreported.
[0066] Cytotoxicity studies of complexes 1, 2, and 3 were
investigated on MCF-7 cells using MTT
(MTT=(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assay methodology. The cell viability was found to be .gtoreq.85%
after incubation with 300 .mu.M of 1, 2, and 3 for 24 h. Thus, MTT
assay confirmed that the insignificant toxicity of all three
complexes towards MCF-7 cell lines (FIG. 13). Evaluated IC50 values
were found to be .gtoreq.300 .mu.M.
[0067] In the light of the negligible toxicity towards MCF-7 cells,
the application of the above complexes as potential as imaging
agents was explored in live MCF-7 cells by incubating the cells
separately with 50 .mu.M of 1, 2, and 3 at 37.degree. C. A close
comparison of the confocal laser scanning microscopic (CLSM) images
as well as the images of the co-staining experiments with
well-known ER staining agent clearly concludes that intracellular
emission for 1 and 2 were found to be exactly superimposed with
those for ER-Tracker.TM. Green (FIG. 19A) Also, distinct emission
of 1 and 2 was also observed from the cell membrane, indicating
that the cell membrane was also a target for the complexes 1 and 2.
Identical studies with complex 3 did not show any such specificity
towards lipid dense regions like endoplasmic reticulum (ER) or cell
membrane. Also, the extent of cellular uptake for complex 3 was
found to be less as compared to the complexes 1 and 2. The enlarged
confocal images for complexes 1 and 2 (FIG. 19C) reveals dot-like
structures with red fluorescence in bright-field microscopy, a
pattern that is observed earlier for localization of lipophilic
reagents at the cell membrane, which are also observed to be
scattered in the cytoplasm suggests that the complexes were also
sequestered in cytoplasmic vacuoles.
[0068] Partition coefficients (log P) for these three complexes
were evaluated by shake-flask method and these were correlated to
the lipophilicity of respective complexes. Lipophilicity of the
complexes 1 (log P=-0.85), 2 (log P=-0.50), and 3 (log P=-1.1) were
evaluated. These data clearly reveal that log P is the highest for
complex 2 followed by 1 and 3 as shown in (table 2).
[0069] In another embodiment, co-staining experiments were
performed with DAPI, a commonly used commercial nuclear staining
agent. For cells treated with complexes 1 and 2, CLSM images were
recorded following excitation with 442 nm laser and intra cellular
emission of respective complexes were observed at 620 nm.
Superimposed fluorescence intensity profile plots (FIG. 19B) of the
intracellular emission signals of DAPI with those of complexes 1 or
2, confirmed that both complexes were as efficient as DAPI in
staining the nucleus of the MCF-7 cells.
[0070] However, complex 3 resulted in non-specific staining of
cellular compartments in fixed cells and CLSM images revealed a
non-specific internalization of complex 3 in cytoplasm and nucleus
of fixed and live MCF-7 cells (FIG. 22 a & b). To ensure that
DAPI had no influence in internalization of the complexes 1 and 2,
identical experiments with fixed MCF-7 cells in absence of DAPI
were performed (FIG. 22 c) and a distinct red emission were
observed from the nucleus. Luminescence intensity profile plots
confirmed that the emission was actually from the nucleus.
[0071] Further, in order to check the preferential binding of these
two reagents to nuclear DNA, deoxyribonuclease (DNase I) and
ribonuclease (RNase A) digest experiments were carried out. DNase I
is an enzyme that catalyzes the hydrolytic cleavage of
phosphodiester linkages in the DNA and RNase A is a type of
nuclease that catalyzes the degradation of RNA into smaller
components. DAPI was used for control experiments. Comparison of
the CLSM images of the cells pretreated with DNase I (FIG. 17)
clearly revealed that the intracellular fluorescence intensities of
complex 1 and 2 from the nucleus of MCF-7 cells were significantly
reduced for cells that were pre-treated with DNase I and not for
those pre-treated with RNase A. This further confirms that
complexes 1 and 2 are mainly targeting nuclear DNA in the fixed
cell nucleus.
[0072] In yet another embodiment, photostability of these two
complexes 1 & 2 compared with that for DAPI dye by using
confocal microscopy. The fluorescence intensity of DAPI upon
continuous irradiation in fixed MCF-7 cells (at 359 nm for 300 sec)
was drastically reduced, whereas, fluorescence intensity of
complexes 1 and 2 were only reduced by .about.20% upon exposure at
442 nm for 300 sec, reflecting the photostability of these two
fluorescent complexes 1& 2 as staining reagents.
[0073] Thus, the present invention provides the Ruthenium (II)
polypyridyl complexes of formula I, for use as DNA imaging
agents.
[0074] In a further embodiment, the invention provides method of
imaging DNA in a tumor cell, which method comprises contacting the
Ruthenium (II) polypyridyl complexes of formula I with cell nucleus
to obtain the images of DNA.
[0075] The method according to the invention, the tumor/cancer cell
may be selected from breast cancer; epithelial cancer, lung cancer,
ovarian carcinoma, pancreatic carcinoma, prostate cancer or
colorectal carcinoma.
[0076] In yet another embodiment, the invention provides a
composition comprising Ruthenium (II) polypyridyl complexes of
formula I along with one or more pharmaceutical carriers for use as
DNA imaging agent.
[0077] The following examples, which include preferred embodiments,
will serve to illustrate the practice of this invention, it being
understood that the particulars shown are by way of example and for
purpose of illustrative discussion of preferred embodiments of the
invention.
EXAMPLES
Example 1
Experimental Details
Materials and Methods:
[0078] All chemicals were purchased from Sigma Aldrich unless
otherwise indicated. All solvents were dried and distilled prior to
use following standard procedures. .sup.1H NMR spectra were
recorded on a Bruker 500 MHz FT NMR (model: Advance-DPX 500)
spectrometer at room temperature. ESIMS measurements were carried
out on a Waters QTof-Micro instrument. UV-Vis spectra were obtained
by using a Cary 500 scan UV-Vis spectrometer. The CT-DNA
concentration per nucleotide was determined by absorption
spectroscopy by using the molar absorption coefficient (6600
mol.sup.-1dm.sup.3cm.sup.-1) at 260 nm. The emission spectra were
obtained using Edinburgh instrument Xe-900 spectro fluorometer.
Synthesis
[0079] The ligands L.sub.1 and L.sub.2 were prepared by alkylation
of 4-(bromomethyl)-4'-methyl-2,2'-bipyridine in DMSO directly with
5-fluorouracil at N.sup.1 position to give
5-fluoro-1-((4'-methyl-[2,2'-bipyridin]-4-yl)methyl)pyrimidine-2,4(1H,3H)-
-dione (L.sub.1), and with uracil to give
1-((4'-methyl-[2,2'-bipyridin]-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione(L.-
sub.2). Reaction of these two ligands with
[Ru(bpy).sub.2Cl.sub.2].2H.sub.2O in ethanol for 8 h under reflux
conditions afford a deep orange colored complexes 1 and 2 purified
by column chromatography (silica 100-200 mesh and acetonitrile as
eluent) and characterized using standard analytical techniques.
Ligand L3 (4,4'-dimethyl-2,2'-bipyridine) was obtained from
Sigma-Aldrich and used without further purification for the
synthesis of complex 3.
Example 2
Synthesis of L.sub.1
[0080] A mixture of 5-fluorouracil (0.130 g, 1 mmol),
K.sub.2CO.sub.3 (0.276 g, 2.0 mmol) and KI (ca. 25 mg) in 10 mL of
DMSO was stirred under N.sub.2 for 10 min.
4-(bromomethyl)-4'-methyl-2,2'-bipyridine (0.644 g, 2.45 mmol)
predissolved in DMSO (5 mL) was then slowly added via a syringe and
the resultant chocolate brown mixture was stirred under N.sub.2 at
room temperature for 3 h. Water (100 mL) was then added and the
suspension was extracted with dichloromethane. The collected
organic layers were dried over anhydrous sodium sulphate and the
solvent removed in vacuum to give a half-white solid. The crude was
subjected to the silica column chromatography using dichloromethane
and acetone as solvent mixture 99:1% (v/v). The second spot from
the bottom on the TLC plate was collected as L.sub.1 (0.150 g,
48%). Electron impact (EI) mass spectrum:
m/z=334.93[L.sub.1+Na.sup.+]. .sup.1H NMR (200 MHz,
methanol-d.sub.4) .delta..sub.H 8.89 (1H, s), 8.68 (d, 1H, J=4.8
Hz), 8.54 (d, 1H, J=4.8 Hz), 8.33 (1H, s), 8.25 (1H, s), 7.19 (d,
2H, J=10.3 Hz), 5.77 (d, 1H, J=7.7 Hz), 5.00 (2H, s), 2.45 (1H, s).
.sup.19F NMR (400 MHz, MeOD-d.sub.4) .delta. -74.85 ppm. (Refer
FIG. 3, 4, 5)
Synthesis of L.sub.2
[0081] A mixture of uracil (0.112 g, 1 mmol), K.sub.2CO.sub.3
(0.276 g, 2 mmol) and KI (ca. 25 mg, a catalytic amount) in 10 mL
of DMSO was stirred under N.sub.2 for 10 min.
4-(bromomethyl)-4'-methyl-2,2'-bipyridine (0.644 g, 2.45 mmol)
predissolved in DMSO (5 mL) was then slowly added via a syringe and
the resultant chocolate brown mixture was stirred under N.sub.2 at
room temperature for 3 h. Water (100 mL) was then added and the
suspension was extracted with dichloromethane. The collected
organic layers were dried over anhydrous sodium sulphate and
solvent was removed in vacuum to give a half-white solid. The crude
was subjected to the silica column chromatography using
dichloromethane and acetone as eluent 99:1% (v/v). The second spot
on the TLC plate was collected as L.sub.2 (0.145 g, 49.26%).
Electron impact (EI) mass spectrum: m/z=316.91[L.sub.2+Na.sup.+];
.sup.1H NMR (200 MHz, MeOD) .delta..sub.H 8.65 (d, 1H, J=5.7 Hz),
8.52 (d, 1H, J=4.8 Hz), 8.24-8.18 (2H, m), 7.73 (d, 1H, J=7.9 Hz),
7.38 (d, 1H, J=3.4 Hz), 7.32 (d, 1H, J=6.6 Hz), 5.77 (d, 1H, J=7.9
Hz), 5.10 (2H, s), 2.49 (3H, s). (Refer FIG. 9, 10)
Example 3
Synthesis of [Ru(bpy).sub.2(L.sub.1)](PF.sub.6).sub.2 (1);
[Ru(bpy).sub.2(L.sub.2)](PF.sub.6).sub.2 (2) And
[Ru(bpy).sub.2(L.sub.3)](PF.sub.6).sub.2 (3)
[0082] Complexes 1 2 and 3 were prepared using the reaction of
[Ru(bpy).sub.2Cl.sub.2].sup.2+ (0.145 g, 0.3 mmol) with the
appropriate ligand L.sub.1 (0.112 g, 0.3 mmol) or L.sub.2 (0.105 g,
0.36 mmol) or L.sub.3 (0.05 g, 0.3 mmol) in ethanol under reflux
condition for 8 h. After cooling, addition of saturated aqueous
potassium hexafluorophosphate (KPF.sub.6) solution precipitated out
the complexes as orange red solids. Which were filtered off using
G4 glass cantered crucible. The precipitate washed with Millipore
water (3 mL.times.5) followed by diethyl ether and dried over
P.sub.2O.sub.5 in desiccator. The both compounds were purified by
silica gel (100-200 mesh) column chromatography using acetonitrile
and saturated KPF.sub.6 solution 98:2% (v/v) as eluent.
Characterization of Complex 1
[0083] Yield: (0.165 g, 0.16 mmol) Electron impact (EI) mass
spectrum: m/z for 1.sup.2+=725.1362 found; 725.1357 calculated
[M-2PF.sub.6].sup.+; .delta..sub.H (500 MHz, CD.sub.3CN) 9.52 (1H,
s), 8.51 (5H, d, J=7.4), 8.44 (1H, s), 8.31 (1H, s), 8.07 (5H, t,
J=7.8), 7.73 (5H, t, J=6.6), 7.69 (1H, d, J=5.8), 7.64 (1H, d,
J=6.2), 7.57 (1H, d, J=5.8), 7.46-7.38 (5H, m), 7.32 (1H, d,
J=5.4), 7.27 (1H, d, J=5.6), 5.01 (2H, s), 2.57 (3H, s); .sup.19F
NMR (400 MHz, CD.sub.3CN) .delta. -168.99 ppm. (FIG. 2,6,7,8);
Characterization of Complex 2
[0084] Yield: (0.159 g, 0.16 mmol) Electron impact (EI) mass
spectrum: m/z for 2.sup.2+=707.1448 found, 707.1451 calculated,
[M-2PF.sub.6].sup.+; .delta..sub.H (400 MHz, CDCl.sub.3) 9.26 (1H,
s), 8.52 (4H, d, J=8.0), 8.43 (1H, s), 8.34 (1H, s), 8.10-8.04 (4H,
m), 7.75 (4H, d, J=4.5), 7.69 (1H, d, J=5.8), 7.57 (1H, d, J=5.7),
7.49-7.39 (5H, m), 7.31-7.25 (2H, m), 5.70 (1H, d, J=7.8), 5.05
(2H, s), 2.56 (3H, s). (FIG. 1,2,11,12)
[0085] Crystal structure and refinement details of complex 2 are
provided in table 1.
Characterization of Complex 3
[0086] Yield: (0.151 g, 57%) ESI-MS: m/z for M.sup.2+=299.07.
.sup.1HNMR (200 MHz, CD3CN) 8.58 (2H, s), 8.54 (2H, s), 8.43 (2H,
s), 8.14-8.04 (4H, m), 7.80 (4H, d, J=5.6), 7.60 (2H, d, J=5.8),
7.50-7.40 (4H, m), 7.31-7.26 (2H, m), 2.57 (6H, s). Elemental
analysis (as chloride salt). Calcd: C, 57.49; H, 4.22; N, 12.57.
Found: C, 57.4; H, 4.2; N, 12.48
Example 4
Single Crystal X-Ray Diffraction Studies and Crystal Structures of
Complex 2
[0087] As-synthesized crystal of complex 2 was coated with
paratone-N and placed on top of a nylon cryoloop (Hampton research)
and then mounted in the diffractometer. The data collection was
done at 298 K. The crystal was mounted on a Super Nova Dual source
X-ray diffractometer system (Agilent Technologies) equipped with a
CCD area detector and operated at 250 W power (50 kV, 0.8 mA) to
generate Mo K.alpha. radiation (.lamda.=0.71073 .ANG.) and Cu
K.alpha. radiation (.lamda.=1.54178 .ANG.) at 298 K. Initial scans
of each specimen were performed to obtain preliminary unit cell
parameters and to assess the mosaicity (breadth of spots between
frames) of the crystal to select the required frame width for data
collection. CrysAlis.sup.Pro program software was used suite to
carry out overlapping .phi. and .omega. scans at detector
(2.theta.) settings (2.theta.=28). Following data collection,
reflections were sampled from all regions of the Ewald sphere to
redetermine unit cell parameters for data integration. In no data
collection was evidence for crystal decay encountered. Following
exhaustive review of collected frames the resolution of the dataset
was judged. Data were integrated using CrysAlis.sup.Pro software
with a narrow frame algorithm. Data were subsequently corrected for
absorption by the program SCALE3 ABSPACK scaling algorithm.
[0088] These structures were solved by direct method and refined
using the SHELXTL 97 software suite. Atoms were located from
iterative examination of difference F-maps following least squares
refinements of the earlier models. Final model was refined
anisotropically (if the number of data permitted) until full
convergence was achieved. Hydrogen atoms were placed in calculated
positions (C--H=0.93 .ANG.) and included as riding atoms with
isotropic displacement parameters 1.2-1.5 times Ueq of the attached
C atoms. Highly porous crystals that contain solvent-filled pores
often yield raw data where observed strong (high intensity)
scattering becomes limited to .about.1.0 .ANG. at best, with higher
resolution data present at low intensity. The structure was
examined using the ADSYM subroutine of PLATON to assure that no
additional symmetry could be applied to the models. The ellipsoids
in ORTEP diagrams are displayed at the 50% probability level (FIG.
1).
Example 5
Photophysical Studies
[0089] UV-Vis spectra were obtained by using a Cary 500 scan
UV-Vis-NIR spectrometer. Room temperature emission spectrum was
obtained using an Edinburgh instrument Xe-900 spectro fluorometer.
The fluorescence quantum yields, .PHI..sub.f were estimated by
using equation 1 in water medium using the integrated emission
intensity of Ru(bpy).sub.3Cl.sub.2 (.PHI..sub.f=0.042 in H.sub.2O
at RT) as a reference. (FIG. 20)
Lifetime Measurements
[0090] Luminescent lifetimes were obtained using a Horiba TCSPC
(Time Correlated Single Photon Counting) system exciting at 443 nm.
10,000 counts were collected for each lifetime measurement and all
measurements were performed in triplicate using DAS software to
confirm results. The calculation of the luminescent lifetimes was
performed by fitting an exponential decay function to each decay
plot to extract the lifetime information using DAS6 fluorescence
decay analysis software (FIG. 2).
Partition Coefficient Measurements:
[0091] n-Octanol saturated water and water saturated octanol was
obtained using Millipore water stirred with n-octanol for 24 h
before the two layers were separated by centrifugation (3000 rpm, 5
min). The chloride salts of complex 1 and 2 were dissolved in
n-octanol saturated water giving concentrations ranging from 0.5 to
3.0 mmol. This was then mixed with water saturated n-octanol in the
ratio of 1:1 (v/v). Resulting solvent mixtures were vertexed for 30
min at room temperature, and then were subjected to centrifugation
(3000 rpm, 5 min) to get two distinct separate layers. Samples from
each layer were obtained using a fine-gauge needle and the
absorbance of respective complex in each phase determined using
UV-Vis spectroscopy.
Isothermal Titration Calorimetry (ITC) Studies:
[0092] ITC experiments were performed with the Microcal iTC200.
CT-DNA (0.1 mmol) and complexes 1 (5 mmol), 2 (5 mmol) and 3 (5
mmol) were used for these experiments. All titrations were
conducted in Tris-HCl buffer (5 mmol Tris and 25 mmol NaCl), pH=7.4
at 25.degree. C. In each titration CT-DNA was loaded into the cell
and 1 or 2 or 3 were taken into the syringe. Aliquot of 2 .mu.L of
compounds were injected into the cell containing DNA. In each
experiment, the raw isotherms were corrected for heat of dilution
by subtracting the isotherms representing the compounds injected
into the Tris-HCl buffer. The resulting isotherms were fitted with
the one set of site binding model provided with Microcal iTC200.
(Refer FIG. 21)
Cell Viability Assays:
[0093] Cell cultures were treated with 0-350 .mu.M solutions of 1,
2 (final medium composition=90% cell media, 10% PBS) in triplicate
for 24 h. After incubation 5 .mu.l of MTT reagent was added and
incubated for 4 hrs. MTT (thiazolyl blue tetrazolium bromide)
dissolved in serum-free media. After 4 h incubation the media was
removed and the formazan product was eluted using isopropanol and
the absorbance at 540 nm quantified by plate reader. An average
absorbance for each concentration was obtained and the metabolic
activity of the cell population was determined as a percentage of
untreated negative control. (FIG. 13)
Microscopy:
[0094] MCF-7 and HEK293T cells were cultured in DMEM respectively
supplemented with 10% FBS and penicillin/streptomycin. Cell lines
were maintained at 37.degree. C. in an atmosphere of 5% CO.sub.2
and routinely sub-cultured. For CLSM, cell cultures were grown on 6
well plate with coverslips, after 24 hrs of incubation the cells
were treated with solutions of 1 or 2 (20 .mu.M,) in serum
containing media and incubated for 2 h. After incubation media was
removed and cells were washed with 1.times.PBS buffer and fixed
them using paraformaldehyde and stained for confocal microscopy.
Nuclear staining was performed by using DAPI. Olympus Fluoview was
used to observe the compound staining Compound was excited at 443
nm and emission was detected at 620 nm wavelength.
[0095] DAPI was excited by using a 405 nm diode laser and emission
detected with a 420-480 nm long-pass band-filter. (FIG. 14, 15, 16,
17)
Cellular Uptake & Quantification of 1, 2, and 3 by (MP-AES)
[0096] Cellular accumulation studies for complexes 1, 2, and 3 were
conducted on the MCF-7 cell line. Briefly,
2.5.times.10.sup.5-1.times.10.sup.6 cells were seeded on a
petridish; the metal complexes were then added to give final
concentrations of 50 .mu.M and allowed a further 24 h of drug
exposure at 37.degree. C. After this time, cells were treated with
trypsin, counted using haemocytometer, and cells collected were
digested overnight in concentrated nitric acid (73%) at 60.degree.
C.; Samples were made up to exactly 10 mL using deionized water and
the amount of ruthenium taken up by the cells was estimated by
MP-AES (Microwave Plasma Atomic Emission Spectroscopy), using an
Agilent Technologies instrument (Model No: 4100 MP-AES). The
solvent used for all MP-AES experiments was double deionized water
(DDW). The concentrations used for the calibration curve were in
all cases 0, 5, 7.5, 10 ppm. The isotope detected was .sup.101Ru;
readings were made in duplicate (N.sub.2 gas mode).
[0097] Thus the present inventors have successfully developed novel
Ruthenium (II) polypyridyl complexes (1 and 2) as imaging reagents.
Both the reagents were preferentially localized in lipid dense
regions such as endoplasmic reticulum, cell membrane, and
cytoplasmic vacuoles of live MCF-7 cells, which illustrates the
role of uracil and 5-fluorouracil functionality in achieving
specificity for the lipid dense regions in live cells. Relatively
higher lipophilicity of complex 2 helped in achieving better
cellular internalization. For fixed cell, the lipid layer
disruption helped in explicit localization in cell nucleus through
specific interaction with cellular DNA. Insignificant toxicity,
photo-stability, visible light excitation, good solubility, high
lipophilicity & permeability and large
.quadrature..quadrature.Ss of .about.160 nm being the
characteristics of these two novel complexes enable their
application as an imaging reagent for DNA in live cells.
TABLE-US-00001 Ru accumulation Complex logP ppm/10.sup.6 cells 1
-0.85 1.5 2 -0.50 2.3 3 -1.1 0.8
TABLE-US-00002 TABLE 1 Crystal structure and refinement details of
2 Empirical formula C37H33F12N9O4P2Ru Formula weight 1058.73
Temperature 298 K Wavelength 0.71073 .ANG. Crystal system
monoclinic Space group C 2/c Unit cell dimensions a = 39.898(4)
.ANG. .alpha. = 90.degree. b = 10.0280(8) .ANG. .beta. =
107.degree. c = 22.1207(14) .ANG. .gamma. = 90.degree. Unit cell
volume 8462.4(13) Z 8 Density (calculated) 1.662 mg mm.sup.-3
Absorption coefficient 0.551 F(000) 4256 Crystal size 0.3 .times.
0.2 .times. 0.2 mm.sup.3 Theta range for data collection 2.95 to
29.14 Index ranges -51 <= h <= 53, -13 <= k <= 13, -27
<= l <= 29 Reflections collected 23879 Independent
reflections 22963 Absorption correction Semi-empirical from
equivalents Refinement method Full-matrix least-squares on F.sup.2
Data/restraints/parameters 9749/0/587 Goodness-of-fit on F.sup.2
1.606 Final R indices [I >= 2sigma(I)] R.sub.1 = 0.0812,
wR.sub.2 = 0.1504 R indices (all data) R.sub.1 = 0.1609, wR.sub.2 =
0.1662 Largest diff. peak and hole 0.841 and -0.626 e
.ANG..sup.-3
TABLE-US-00003 TABLE 2 Log P values obtained by using shake-flask
method. Molinspiration software was used for calculation of log p
values for uracil and 5-FU. (http://www.molinspiration.com/
cgi-bin/properties). Compound 1 2 Uracil 5-Fluorouracil LogP -0.99
.+-. 0.05 -1.59 .+-. 0.09 -0.89 -0.58
TABLE-US-00004 TABLE 3 Photophysical properties of the complexes 1,
2, and 3 recorded in pure aqueous medium. .lamda..sub.abs/nm
(.epsilon./10.sup.3M.sup.-1 .lamda..sub.ex max/nm .lamda..sub.em
max/nm Complex cm.sup.-1) (.lamda..sub.em) (.lamda..sub.ex)
.PHI..sub.f .tau. (ns) 1 246 (18.59) 249 617 0.048 336 287 (54.74)
305 455 (9.41) 461 (617) 2 247 (28.08) 232 617 0.050 323 287
(79.35) 293 455 (13.85) 461 (617) 3 246 (18.01) 240 619 0.05 321
290 (55.05) 308 456 (14.60) 459 (619)
TABLE-US-00005 TABLE 4 ITC binding parameters for interaction of 1
and 2 with CT-DNA. Complex 2 1 3 .DELTA.H [Kcal/M.sup.-1] 1.47 .+-.
0.02 -35.5 .+-. 0.41 0.51 .+-. 0.25 -T.DELTA.S 7.47 28.87 5.03
[Kcal/M.sup.-1l] .DELTA.G [Kcal/M.sup.-1] -6.00 .+-. 0.02 -6.68
.+-. 0.41 -4.52 .+-. 0.25 N [bp] 0.98 .+-. 0.01 0.61 .+-. 0.0 0.354
.+-. 0.14 K.sub.a [M.sup.-1] (2.51 .+-. 0.16) 10.sup.4 (7.23 .+-.
0.24)10.sup.4 (2.05 .+-. 0.85)103
ADVANTAGES OF THE INVENTION
[0098] a. Novel complexes [0099] b. Simple & one step synthesis
[0100] c. Complexes are used for cellular DNA imaging. [0101] d.
Complexes are water soluble, non-toxic, good lifetime, and their
fluorescence falls in visible range with large stokes shift.
* * * * *
References